Use of polyunsaturated ketones for the treatment of psoriasis

ABSTRACT

Psoriasis is a common, chronic, inflammatory skin disorder. This invention provides the use of a compound of formula (I) R—CO—X) (Wherein R is a C 16-24  unsaturated hydrocarbon group optionally interrupted by α, β, γ, or δ to the carbonyl group by a heteroatom or group of heteroatoms selected from S, O, N, SO, SO 2  said hydrocarbon group comprising at least 5 non-conjugated double bonds; and X is an electron withdrawing group) in the manufacture of a medicament for the treatment of psoriasis.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.10/502,414, filed Jan. 11, 2005, which is the U.S. National Stage ofInternational Application No. PCT/GB03/00364, filed Jan. 29, 2003, whichclaims priority to GB0202002.2, filed Jan. 29, 2002. The entire contentsof these applications are incorporated herein by reference.

This invention relates to the use of certain polyunsaturated long-chainketones for the treatment of psoriasis and in particular to ketonescarrying electron withdrawing substituents alpha to the carbonylfunctionality in such treatment.

Psoriasis is a common, chronic, inflammatory skin disorder. Psoriatictissue is characterised by chronic inflammation in both epidermis anddermis, the disease being further characterised by hyperplasia ofepidermal keratinocytes, fibroblast activation, alteration of eicosanoidmetabolism, and leukocyte infiltration. Effective treatments forpsoriasis such as cyclosporin A, steroids, methotrexate andphotochemotherapy all have immunosuppressive activity and are thus notideal treatments due to their side effects. Scientists have thereforepursued other potential treatments for this disease.

It has been observed that psoriatic tissue exhibits elevated levels ofarachidonic acid and eicosanoids. This suggests that phospholipase A₂(PLA₂) may be involved in the pathogenesis of psoriasis. Thephospholipases are a group of enzymes that release unsaturated fattyacids from the sn2 position of membrane phospholipids. Once released,the fatty acids are converted by various enzymes into biologically veryimportant signalling molecules. Release of arachidonate initiates thearachidonate cascade leading to the synthesis of eicosanoids such asprostaglandins. Eicosanoids are important in a variety of physiologicalprocesses and play a central role in inflammation. In Inflammation, Vol.18, No. 1 1994, Andersen et al identify the presence of certainphospholipases in psoriatic human skin.

It is therefore believed that inhibition of phospholipase enzymes shouldhave potential in curing some of the inflammatory symptoms, includingepidermal hyperproliferation due to increased leukotriene production,related to eicosanoid production and cell activation in both epidermisand dermis in psoriasis.

In J. Chem. Soc. Perkin Trans. 1, 2000, 2271-2276 several structurallydifferent compounds are reported as inhibitors of cPLA₂ in vitro. Thecompounds tested were based around(all-Z)-eicosa-5,8,11,14,17-pentaenoic acid (EPA) and(all-Z)-docosa-4,7,10,13,16,19-hexaenoic acid (DHA). The paper suggeststhat preliminary studies show that in vitro the compounds are active asenzyme inhibitors.

The compounds in J. Chem. Soc. Perkin Trans. 1, 2000, 2271-2276 havehowever not been tested in vivo and there is thus no way of predictingtheir in vivo effects. In addition, when devising a treatment for adisease it is necessary to ensure selectivity. There are a very largenumber of phospholipase enzymes known and more enzymes of this type arebeing discovered as medical science develops. Since phospholipasescontrol a wide variety of different intracellular functions it isnecessary to develop inhibitors of these enzymes that are selective forthe particular phospholipase whose activity is to be altered. Compoundswhich inhibit a large number of phospholipase enzymes are of littlecommercial interest since the benefits of a desired enzymic inhibitionwill be opposed by the presence of many unwanted and potentiallydangerous side effects caused by unwanted enzyme inhibitions. Thereremains a need therefore, to provide highly selective inhibitors ofphospholipase enzymes.

The present inventors have surprisingly found that compounds of somewhatsimilar structure or the same structure as those identified in thePerkin Transactions paper are selective for IVa PLA enzymes and aretherefore ideal candidates for the treatment of psoriasis in the absenceof side effects. Given that there are a total of 23 enzymes in thephospholipase group and each enzyme fulfils a different physiologicaland pathological function this is surprising. Moreover, the compounds ofthe invention have surprisingly been found to be particularly potent inreducing eicosanoid production, by for example, the inhibition of groupIVa PLA₂.

Thus, viewed from one aspect the invention provides the use of acompound of formula (I)R—C—X  (I)

(wherein R is a C₁₆₋₂₄ unsaturated hydrocarbon group optionallyinterrupted α, β, γ, or δ to the carbonyl group by a heteroatom or groupof heteroatoms selected from S, O, N, SO, SO₂, said hydrocarbon groupcomprising at least 5 non-conjugated double bonds; and

X is an electron withdrawing group) for the manufacture of a medicamentfor the treatment of psoriasis.

Viewed from another aspect the invention provides a method of treatingpsoriasis comprising administering to an animal, preferably a mammal,e.g. human, an effective amount of a compound of formula (I) ashereinbefore described.

Viewed from another aspect the invention provides use of a compound offormula (I) as hereinbefore described for use in the manufacture of amedicament for inhibiting the enzyme IVa PLA₂.

Viewed from yet another aspect, the invention provides a pharmaceuticalcomposition comprising a compound of formula (I) as hereinbeforedescribed.

The group R preferably comprises 5 to 7 double bonds, preferably 5 or 6double bonds, e.g. 5 double bonds which should be non-conjugated. It isalso preferred if the double bonds do not conjugate with the carbonylfunctionality.

The double bonds present in the group R may be in the cis or transconfiguration however, it is preferred if the majority of the doublebonds present (i.e. at least 50%) are in the cis configuration. Infurther advantageous embodiments all the double bonds in the group R arein the cis configuration or all double bonds are in the cisconfiguration except the double bond nearest the carbonyl group whichmay be in the trans configuration.

The group R may have between 16 and 24 carbon atoms, preferably 19 to 21carbon atoms.

The group R may carry a heteroatom or group of heteroatoms positioned α,β, γ, or δ to the carbonyl, preferably β or γ to the carbonyl.Preferably the heteroatom is O or S or a sulphur derivative such as SO.

Specifically preferred RCOX groups are those of formula

The R group may carry up to three substituents selected from halo orC₁₋₆-alkyl. If present the substituents are preferably non-polar, andsmall, e.g. a methyl group. It is preferred however, if the R groupremains unsubstituted.

The group X is an electron withdrawing group. Suitable groups in thisregard include O—C₁₋₆ alkyl, CN, CO₂-C₁₋₆ alkyl, phenyl, CHal₃, CHal₂H,CHalH₂ wherein Hal represents a halogen, e.g. fluorine, chlorine,bromine or iodine, preferably fluorine.

In a preferred embodiment the electron withdrawing group is CHal₃,especially CF₃.

Highly preferred compounds for use in the invention are EPACOCF₃,EPASCOCF₃ and AKH 217 as depicted below.

Compounds of formula (I) may be manufactured using known chemicalsynthetic routes. It is convenient to begin synthesis from thecommercially available compounds arachidonic acid, EPA or DHA.Conversion of the acid functionality of these compounds into, forexample a —COCF₃ group can be achieved readily, e.g. by converting thecarboxylic acid into its corresponding acid chloride and reacting thesame with trifluoroacetic anhydride in the presence of pyridine.

Introduction of a heteroatom into the carbon chain is also achievedreadily. Conveniently, for example, the starting acid is reduced to analcohol and, if required, converted to the corresponding thiol. Thenucleophilic thiol may then be reacted with a group such as BrCH₂COCF₃thereby introducing the carbonyl and electron withdrawing species.Complete synthetic protocols may be found in J. Chem. Soc., Perkin Trans1, 2000, 2271-2276 or J. Immunol., 1998, 161, 3421.

The compounds of formula (I) may be formulated into medicaments usingconventional techniques well known to the skilled pharmaceuticalchemist. Thus, the compounds may be formulated with well knownexcipients or pharmaceutical carriers.

The medicaments of the invention may also comprise other conventionaladditives such as antioxidants, preservatives, colouring, flavouringetc.

The medicaments of the invention may be formulated as tablets, pills,powder, capsules, emulsions but are preferably in the form of creams orointments. The mode of administration may be any known mode, such asoral, nasal, transmucosal, parenteral, topical, intradermal etc.However, it is advantageous if the medicament is applied topically, i.e.directly to the infected part of the human skin.

The amount of the medicament required to effect a successful treatmentwill, of course, depend on the patient and on the severity of thepsoriasis. The dose will be readily determined by the skilled chemist.

The compounds of the invention may be used to treat psoriasis incombination with other known pharmaceuticals for said purpose and thisforms a further aspect of the invention.

The invention is described further below with reference to the followingnon-limiting examples and figures.

FIG. 1 shows the relative inhibition of IVa PLA₂ enzyme activity for anumber of compounds of the invention in comparison to commercialcompounds. Recombinant IVa PLA₂enzyme was preincubated with inhibitor (5μM) for 10 minutes and then assayed in the mixed-micelle enzyme activityassay. The control was not pretreated with inhibitor. Results are givenas % of control and are mean of duplicate determinations from 1 out of 4representative experiments.

FIG. 2 shows concentration dependent inhibition of IVa PLA₂in themixed-micelle enzyme activity assay. Increasing inhibition of IVa PLA₂byEPACOCF₃, EPASCOCF₃ and AACOCF₃ are shown in Figure B, and increasinginhibition of IVa PLA₂by EPACH(OH)COCF₃, DHACOCF₃ and

MAFP are shown in Figure A. Results are given as % of control and aremean of duplicate determinations from one out of 2 to 4 representativeexperiments.

FIG. 3A shows that calcium ionophore A₂₃₁₈₇ stimulates extracellularrelease of [3H]-labelled lipid in a concentration-dependent manner.HaCaT cells were treated with A₂₃₁₈₇ for 1 hour, arachidonic acid andeicosanoids were extracted from cell media using Bond Elut C18 columnsand contents of [3H]-labelled lipids in media were determined byscintillation counting. Each column in the figure represents the averageof triplicate determinations from one out of 3 representativeexperiments.

FIG. 3B shows that calcium ionophore A₂₃₁₈₇ dosedependently alsostimulates production of prostaglandin E₂ (PGE₂), a product ofcyclooxygenase modification of arachidonic acid (AA). PGE₂ accumulationin medium was assayed by PGE₂ enzyme immunoassay (EIA).

FIG. 4 shows that the fatty acid derivative EPACOCF₃ dosedependently ismore powerful than the commercially available cyclooxygenase 2-selectiveinhibitor NS-398 in inhibiting A₂₃₁₈₇-induced PGE₂ release inLPS-stimulated HaCat cells (IC₅₀ values 180 nM and 240 nM,respectively).

FIG. 5 shows that TNFα or IL-1β stimulated NF-kB activation isdose-dependently inhibited by AKH217 (91% and 81% respectively).

MATERIALS AND METHODS

Materials

Calcium ionophore A₂₃₁₀₇, Sigmacoat,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) andbovine serum albumin were obtained from Sigma (St. Louis, Mo., USA).Phosphatidylcholine, 1-stearoyl-2-arachidonyl and [3H] arachidonic arefrom Amersham (Buckinghamshire, UK). Aluminium sheets silica gel 60,ethyl acetate, iso-octane and acetic acid were purchased from Merck(Darmstadt, Germany). TNFα was a generous gift from Professor TerjeEspevik, Norwegian University of Science and Technology, NTNU and IL-1βwas purchased from Roche Molecular Biochemicals.

AACOCF₃is from BIOMOL (Plymouth Meeting, Pa., USA), and MAFP is fromCayman (Ann Arbor, Mich., USA). All fatty acid compounds were storedunder N₂ at −80° C.

Cell Culture

The spontaneously immortalized human skin keratmocyte cell line HaCaTwas kindly provided by Prof. N. E. Fusenig (Heidelberg, Germany). Cellswere grown in Dulbecco's Modified Eagle's Medium (DMEM) with 1 gglucose/1 (Gibco BRL, Life Technologies Ltd, Paisley, Scotland),supplemented with 5% fetal calf serum (FCS) (HyClone Laboratories, Inc.,Utah, USA), 0.3 mg/ml L-glutamine (Sigma Chemical Company, St. Louis,Mo., USA), 0.1 mg/ml gentamicine (Sigma) and 1 μg/m1 fungizone (Gibco).Confluent cells were stimulated with A₂₃₁₈₇, Il-1β (10 mg/ml) or TNFα(10 mg/ml) in 0.5% (v/v) FCS for 1 hour before harvesting. Passages40-80 of the cells were used. Generation of HaCat transfectantsexpressing luciferase under strict control of transcription factor NF-kBis described elsewhere (Anthonsen et al, J. Biol. Chem. 2001, 276,30527). The reporter plasmid pBIIX contains two copies of the HIV NF-kBsequence cloned upstream of the mouse fos promoter driving expression ofthe Photinus pyralis luciferase gene.

EIA detection was performed according to manufacturer description,Cayman Chemical, Michigan, USA. A dilution of 1:10 was used for PGE₂measurement. Microplate Manager Software (Bio-Rad Laboratory) calculatedsample data.

Luciferase Assay

Cells were seeded in 24-round multiwell plates (2.8×10⁵ cells/well).Treated cells were washed two times with phosphate-buffered saline andlysed, and luciferase activities were determined using the LuciferaseReporter Assay system (Promega) and Turner Luminometer model TD-20/20(Turner Designs) as described by the manufacturer.

Synthesis of (all-Z)-eicosa-5,8,11,14,17-pentaenoic acid (EPA) and(all-Z)-docosa-4,7,10,13,16,19-hexaenoic acid (DHA) derivatives

The derivatives used in the enzyme assays are shown below. EPACOCF₃ wasprepared as described in J.

Immunol., 1998, 161, 3421. AACOCF3 and MAFP were bought from suppliersas mentioned above. The remaining derivatives were prepared as describedin J. Chem. Soc. Perkin Trans. 1, 2000, 2271-2276.

Mixed-micelle Assay of cPLA₂Activity

Sources of IV PLA₂enzyme activity were insect cells over expressingrecombinant human IV PLA₂ (10 μg IV PLA₂ protein/10⁶ cells; Bac PAKBaculovirus expression system; CLONTECH Laboratories, Palo Alto, Calif.,USA). Cytosolic fractions of insect cells were prepared as described inSchalkwijk et al (1992) Eur. J. Biochem. 210, 169-176. The proteincontents of the cytosolic fractions were measured with Bio-Rad proteinassay (Bio-Rad Laboratories GmbH, Munich, Germany) using bovine serumalbumin as standard. The inhibitory derivatives were added 10 minutesprior to substrate addition. The preincubation of inhibitors wasperformed at room temperature. IV PLA₂ enzyme activity was analyzedusing [14C]-L-3 Phosphatidylcholine, 1-stearoyl-2-arachidonyl assubstrate according to Wijkander et. al. (Eur. J. Biochem. 202, 873-880,1991). After 30 min the reaction was stopped and centrifuged, the CHCl₃phase was evaporated with N₂ gas to dryness and then resuspended inCHCl₃:MeOH (9:1, v/v). Thin layer chromatography (TLC) separated freearachidonic acid from phospholipids on aluminum sheets silica gel 60developed with ethyl acetate: iso-octane:acetic acid: water(55:75:8:100, v/v/v/v) (Gronnich et al, J. Clin. Invest., 93, 1224-1233,1994). Phosphor-Imager quantified free arachidonic acid andphospholipids, and IV PLA₂ activity was expressed as decreasedarachidonic acid release by enzyme incubated with inhibitor compared tono inhibitor.

Arachidonic acid and eicosanoid detection

Confluent cells were labelled with 1 μCi/ml [³H] arachidonic acid inmedia supplemented with 0.5% FCS 24 hours before cell induction andinhibition. About 90% of the radioactive arachidonic acid wereincorporated in the cell membranes. Extracellular [³H] arachidonic acidwas removed by washing the cells 3 times in media. The HaCaT cells werethen preincubated with inhibitor for 1 hour and stimulated with calciumionophore for 1 hour. The cell media were collected and cleared bycentrifugation. Arachidonic acid and eicosanoids were extracted frommedia using Bond Elut C18 octadecyl columns (500 mg) (Varian SPP, HarborCity, Calif.) as described by Powell, Anal. Biochem. 164, 117-131, 1987;with modifications previously described Brekke, Cytokine, 4, 269-280,1992. The samples were collected in glass tubes precoated withSigmacoat. The ethyl acetate solution of samples were completely driedwith N₂, redissolved in 0.5 ml fresh ethyl acetate and aliquots of 50 μl(triplicates) samples were subjected to liquid β-scintillation counting(Beckman L S 1701) in 5 ml Ready Protein liquid (Beckman).

The amount of PGE₂in cell culture media from calcium ionophorestimulated HaCaT cells was measured using an enzyme immunoassay (EIA;Cayman). The assay is based on the competition between free PGE₂and aPGE₂-acetylcholinesterase for a limited amount of PGE₂ monoclonalantibody. The media were diluted 1:10 before analyzing of the PGE₂contents. Microplate Manager Software (Bio-Rad Laboratory) calculatedthe sample data.

MTT Assay

Confluent cells were pretreated with inhibitors in serum free medium for1 hour, and then treated with stimulating agent for 1 hour. Conversionof substrate [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide] was measured as optical density at 580 nm after 4 hoursaccording to Mosmann [Mosmann T, J. Immunol. Methods 65, 55-63, 1983].For each concentration of inhibitor nine parallels were measured.

Results

IV PLA₂Enzyme Activity

In order to investigate the action of the fatty acid derivatives asinhibitors of IV PLA₂, we measured the IV PLA₂ activity in the mixedmicelle assay with recombinant IV PLA₂as enzyme source, as described inmaterials and methods. The synthetic fatty acid derivatives we made arelisted above, together with the commercial available inhibitors, whichwe used for comparison. EPACOCF₃, EPASCOCF₃and AKH-217 seem to have thesame potency as IV PLA₂ inhibitors as AACOCF₃ (i.e. 75-80% inhibition)(FIG. 1). MAFP and DHACOCF₃were poorer IV PLA₂ inhibitors (50% and 30%inhibition respectively). The compound EPACH(OH)CF₃ was also testedwhich results in severe attenuation of inhibitory effect (10%inhibition). EPACOCH₃ was made as a control compound with methyl insteadof the trifluoromethyl (CF₃) group. EPACOCH₃ showed no inhibition (FIG.1).

The IC₅₀ values of EPACOCF₃, EPASCOCF₃ and AACOCF₃ were measured to be2.9±1.9, 3.5±0 μM and 5.8±1.9 μM respectively (FIG. 2B). While the IC₅₀values of DHACOCF₃, MAFP and EPACH(OH)CF₃were determined to be 21.3±1.5μM, 24±1.4 μand 43±7.1 μM respectively (FIG. 2A).

Kinetic studies with the inhibitors in the mixed micelle assay wereperformed in order to see if the time course was linear. A peak wasachieved in two minutes (results not shown), indicating that theinhibitors are very fast acting.

In summary EPACOCF₃, EPASCOCF₃and AKH217 seem to have similar or perhapsslightly higher potency as the commercially available compound AACOCF₃ininhibiting IVa PLA₂.

Arachidonic Acid and Eicosanoid Detection.

In order to evaluate the effect of EPA and DHA derivatives in a morebiological system, we utilized the HaCaT cells as a model system[Sjursen et al, Cytokine, 12, 8, 1189-1194, 2000]. The calcium ionophoreA₂₃₁₈₇ has been shown to induce arachidonic acid release in many celltypes, probably by increasing the intracellular Ca²⁺-concentration andthereby inducing the association of cPLA₂with cellular membranes [Kramerand Sharp, FEES Lett, 410, 49-53, 1997]. In HaCaT cell, the ionophoreinduced a dose response release of [3H]-labelled arachidonic acid (FIG.3A).

Concentrations higher than 10 μM of A₂₃₁₈₇ were toxic as determined byMTT assay.

The next step in evaluating our synthetic fatty acid inhibitors was toexamine their ability to reduce the extracellular release of PGE₂ inresponse to A₂₃₁₈₇ in HaCaT cells. Before the cell experiments wereperformed, we evaluated the toxicity of the inhibitors. MTT assay showedthat concentrations of 25 μM and higher of the fatty acid compounds aretoxic to HaCaT cells (results not shown).

HaCat cells upregulate expression of cyclooxygenase 2 message whentreated with LPS (200 ng/ml, 5% human serum) for 30 min. (unpublishedresults). Upon ionophore stimulation for one hour, PGE₂ accumulates inmedium (FIG. 3B). The fatty acid derivative EPACOCF₃ was compared toNS-398, a cox-2 selective inhibitor, in its potency to reduce PGE₂production by preventing release of its precursor AA. HaCat cells werepretreated with LPS (200 ng/ml, 5% human serum) for 30 min., treatedwith either inhibitors at different concentrations (0.2-25 μM) foranother 30 min and stimulated with A₂₃₁₈₇, 30 min. Finally, PGE₂ EIA wasperformed on cell media. The dose-response curve was constructed withrespect to percent PGE₂ production without inhibitor. As can be observedin FIG. 4, EPACOCF₃ is much more potent than NS-398 in inhibiting PGE₂production (IC₅₀ values of 180 nM and 240 nM, respectively), andindicates that substrate deprivation may be more powerful than activityinhibition for the cyclooxygenase.

In order to determine if inhibition of IVa PLA₂ has any biologicconsequence, HaCaT cells were stimulated with the proinflammatorycytokines IL-1β or TNFα. As a measure of inflammation, activation of thetranscription factor NF-kB was analysed. We have shown earlier that TNFαor IL-1β activates NF-kB in HaCat cells (Thommesen et al, J. Immunol.,1998, 161, 3421). NF-kB activation was analysed as luciferaseexpression. Treatment of the stably transfected HaCat-pBIIX cells withTNFα or IL-1β. for 1 h enhanced NF-kB-dependent expression (not shown).In the presence of inhibitors AKH217, IL-1β stimulated luciferaseexpression was dose-dependently inhibited by 81%. TNF60 stimulated NF-kBactivation was inhibited does-dependently AKH217 by 91% (FIG. 5) thusconfirming that our synthetic fatty acid inhibitors may be useful ininhibiting inflammatory responses.

The invention claimed is:
 1. A pharmaceutical composition comprising acompound of formula (I)R—CO—X  (I), wherein R is a C₁₆₋₂₄ unsaturated hydrocarbon groupinterrupted β or γ to the carbonyl group by one or more heteroatomsselected from the group consisting of S, 0, N, SO, and SO₂, saidhydrocarbon group comprising at least 5 non-conjugated double bonds; andX is an electron withdrawing group, wherein the pharmaceuticalcomposition is a cream or ointment formulated for topical or intradermaluse; wherein the compound of Formula (I) is